This proposal examines the very basic cell biological questions of how eukaryotic cells sort proteins within the Golgi body and separate out those proteins destined for the lysosome, and ultimately deliver these proteins to the lysosome. The simple eukaryote yeast will be used as a model system, since the secretory pathway in yeast has been found to be strikingly similar to that observed in higher eukaryotic organisms. Yeast offers the advantage that it is readily amenable to genetic and biochemical analysis. Yeast mutants will be obtained that are defective in the sorting, packaging and transport of glycoproteins to the lysosome-like vacuole. These mutants will be analyzed to determine precisely what step in the vacuole assembly pathway is defective. In conjunction with studying the components of the vacuole assembly pathway, the recognition of sorting signals present on the vacuolar proteins that target their localization will be investigated. These studies should provide molecular information on the nature of eukaryotic protein recognition signals and how these signals control the intracellular localization of proteins. The diseases pseudo Hurler polydystropy and l-cell disease arise because of genetic defects preventing the correct localization of lysosomal enzymes. In addition, defects in the basic process of secretion may be relevant to the disease cystic fibrosis.